Nicotinamide nucleotides play a major role in biological oxidations; reductive synthesis of carbohydrates, lipids and amino acids; the various monooxygenase reactions involved in detoxification and steroid hydroxylation reactions; energy production; and metabolic regulation. Nicotinamide nucleotide transhydrogenases are potential control devices for maintaining the cellular balance of NAD(H) and NADP(H), and thereby contributing to the regulation of various metabolic reactions linked to these nucleotides. The mitochondrial transhydrogenase system is of particular interest, because the equilibrium of the mitochondrial nicotinamide nucleotide pool can be shifted in favor of NADPH production in an endergonic reaction, which utilizes stoichiometric amounts of ATP. The objectives of the program are (1) to study the molecular mechanisms of nonenergy-linked (NAD(P)H yields NAD) and energy-linked (NAD(P)H yields NADP) transhydrogenation; (2) to isolate and characterize the enzyme(s) involved; (3) to investigate the mechanisms of energy transfer to, and utilization by, the transhydrogenase enzyme; and (4) as a long term goal to relate these findings to studies on (a) the general mechanism of energy conservation, transduction and transfer by mitochondria, and (b) the physiological role of the mitochondrial transhydrogenase reaction. Our work to date has shown that the purified and soluble mitochondrial NADH dehydrogenase is capable of NADPH oxidation and NAD(P)H yields NAD transhydrogenation, and that NADP reduction by either NADH or NADPH is an energy-linked process in mitochondria. The reason for the latter has been explained on the basis of possible electrostatic interaction of the negatively charged 2'-phosphate of NADP with its nicotinamide C-4, which carries a partial positive charge, thus contributing to stabilization of the folded structure of NADP in solution. We have also shown that the transhydrogenase enzyme contains an essential arginyl residue, apparently for specific recognition and binding of the 2'-phosphate of NADP(H).